Investigating the Biosynthesis of the Ambruticins: Total Synthesis and Epoxidation of Intermediate Ambruticin J

The ambruticins are a family of polyketide natural products which display potent antifungal activity. The biosynthesis of the ambruticins occurs on a Type I polyketide synthase (PKS), with two of the three rings being formed on the PKS. The third ring, a tetrahydropyran, is thought to be formed through post-PKS modifications via an epoxidation-cyclization cascade. While this type of cascade is common in biosyntheses, usually two enzymes are required; however, the amb gene cluster encodes only an epoxidase and no epoxide hydrolase. Therefore, the mechanism of tetrahydropyran ring formation and control is currently unknown.

The first chapter of this thesis addresses previous work focused on the ambruticins. Several members of this class have been isolated and identified, and all have displayed promising antifungal activity. The bioactivity spectrum of ambruticin S led to extensive in vitro and in vivo testing against a range of clinically relevant fungal pathogens as well as a number of structure-activity relationship studies, conducted with the goal of creating an ambruticin derivative with improved bioactivity. In addition, several total syntheses of ambruticin S have been completed. Finally, chapter 1 addresses the biosynthesis of the ambruticins, which has been partially explored by several groups. However, little exploration of the methodology of tetrahydropyran ring formation has been carried out.

The second chapter of this work discusses the total synthesis of the putative biosynthetic intermediate ambruticin J, in which the tetrahydropyran ring has not yet been formed. This intermediate is thought to be the product of the ambruticin PKS and is likely the substrate for an epoxidation-cyclization cascade which forms the THP ring of the isolated ambruticins. We have completed a total synthesis of this molecule, providing access to ambruticin J for epoxidation-cyclization studies and synthetically confirming the proposed structure.

The third chapter of this work provides an overview of known biosynthetic epoxidation-cyclization cascades and addresses the factors which control regioselectivity of epoxide-opening in ring formation reactions. Finally, chapter four discusses the development of epoxidation-cyclization conditions for ambruticin J and their application to ambruticin J model systems. This chapter also discusses the implications for ambruticin biosynthesis arising from these results.